Increases in astrocyte Ca(2+) have been suggested to evoke gliotransmitter release, however, the mechanism of release, the identity of such transmitter(s), and even whether and when such release occurs, are controversial, largely due to the lack of a method for selective and reproducible stimulation of electrically silent astrocytes. Here we show that photoactivation of the light-gated Ca(2+)-permeable ionotropic GluR6 glutamate receptor (LiGluR), and to a lesser extent the new Ca(2+)-translocating channelrhodopsin CatCh, evokes more reliable Ca(2+) elevation than the mutant channelrhodopsin 2, ChR2(H134R) in cultured cortical astrocytes. We used evanescent-field excitation for near-membrane Ca(2+) imaging, and epifluorescence to activate and inactivate LiGluR. By alternating activation and inactivation light pulses, the LiGluR-evoked Ca(2+) rises could be graded in amplitude and duration. The optical stimulation of LiGluR-expressing astrocytes evoked probabilistic glutamate-mediated signalling to adjacent LiGluR-non-expressing astrocytes. This astrocyte-to-astrocyte signalling was insensitive to the inactivation of vesicular release, hemichannels and glutamate-transporters, and sensitive to anion channel blockers. Our results show that LiGluR is a powerful tool to selectively and reproducibly activate astrocytes.